Heterogeneity of NH+4 transport in mouse inner medullary collecting duct cells

Abstract

Previous studies from our laboratory have demonstrated that NH+4 substitutes for K+ on the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in rat terminal inner medullary collecting duct cells (tIMCD). To examine other NH+4 transport pathways, a transgenic mouse cell line, mIMCD-3, was employed. 86Rb+ was used as a K+ congener to explore NH+4/Rb+ (K+) competition on the extracellular K+ binding site of the Na(+)-K(+)-2Cl- cotransporter and the Na(+)-K(+)-ATPase. Addition of K+ or NH+4 reduced both bumetanide- and ouabain-sensitive Rb+ uptake. This reduction in Rb+ uptake with NH+4 addition was not due to intracellular pH-mediated changes in transporter activity. K+ and NH+4 are competitive inhibitors on both transporters. On the Na(+)-K(+)-2Cl- cotransporter, the Michaelis constant (Km) for K+ was 4.6 +/- 0.5 mM with an inhibitory constant (Ki) for NH+4 of 2.8 mM. In contrast, on the Na(+)-K(+)-ATPase, the apparent affinity for K+ was greater than for NH+4. To test Na(+)-K(+)-2Cl- cotransport-mediated NH+4 flux, bumetanide-sensitive NH+4/Rb+ exchange was measured. Bumetanide-sensitive Rb+ efflux was greater with extracellular K+ or NH+4 present relative to efflux with extracellular N-methyl-D-glucamine. This demonstrates both K+/Rb+ and NH+4/Rb+ countertransport by the Na(+)-K(+)-2Cl- cotransporter. In conclusion, NH+4 is transported in a bumetanide-sensitive Na(+)-NH+4-Cl- mode, and both NH+4 and Rb+ (K+) are competitive inhibitors for the extracellular K+ binding site. However, the kinetics of Na(+)-K(+)-2Cl(-)-mediated NH+4 transport differ from other K+ transport-mediated NH+4 pathways, such as the Na(+)-K(+)-ATPase.

Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct

“…Although the bumetanide concentrations used in this study (100 -300 M) are higher than those used in cultured cells or parotid acinar cells (0.5-10 M) (19), 100 M bumetanide is needed to fully inhibit NKCC1 in rat (13,27) or mouse (29) collecting duct cells. At these higher bumetanide concentrations, K ϩ -Cl Ϫ cotransporters are partly inhibited (16).…”

Rh glycoprotein expression is modulated in pufferfish (Takifugu rubripes) during high environmental ammonia exposure

“…Due to a similar hydrated radius, NH 4 + is known to substitute for K + in the NKCC and the NKA in a variety of different tissues (Kinne et al, 1986;Kurtz and Balaban, 1986;Towle and Holleland, 1987;Wall et al, 1995). NKA has been implicated in the basolateral uptake of ammonia in the collecting duct of the mammalian kidney (Kurtz and Balaban, 1986;Wall and Koger, 1994) and similarly, basolateral gill Na + /NH 4 + exchange via NKA has been suggested from studies on the toadfish (Claiborne et al, 1982;Mallery, 1983).…”

In rat inner medullary collecting duct, NH 4 + uptake by the Na,K-ATPase is increased during hypokalemia

“…tIMCDs were perfused and bathed at 37°C in symmetrical Cl Ϫ -and Na ϩ -free solutions (solution 6) that also contained 10 M Sch-28080, 2 mM BaCl2, 100 M amiloride, 100 M bumetanide, and 5 mM ouabain. These transport inhibitors were selected to inhibit pHi regulatory mechanisms and NH 4 ϩ transport mechanisms (36,37,42,44,45). pHi was clamped at values between 7.0 1 After 3 wk of a K ϩ -restricted diet, intracellular K ϩ concentration falls by 30 mM (3) in rat tIMCD in vivo.…”